In Vitro and In Vivo Assessment of PEGylated PEI for Anti-IL-8/CxCL-1 siRNA Delivery to the Lungs

Alan J Hibbitts,Sally-Ann Cryan,Ronan Macloughlin,James Barlow,Joanne M Ramsey

doi:10.3390/nano10071248

Alan J Hibbitts, Sally-Ann Cryan + Show 3 more

Open Access

https://doi.org/10.3390/nano10071248

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Abstract

Inhalation offers a means of rapid, local delivery of siRNA to treat a range of autoimmune or inflammatory respiratory conditions. This work investigated the potential of a linear 10 kDa Poly(ethylene glycol) (PEG)-modified 25 kDa branched polyethyleneimine (PEI) (PEI-LPEG) to effectively deliver siRNA to airway epithelial cells. Following optimization with anti- glyceraldehyde 3-phosphate dehydrogenase (GAPDH) siRNA, PEI and PEI-LPEG anti-IL8 siRNA nanoparticles were assessed for efficacy using polarised Calu-3 human airway epithelial cells and a twin stage impinger (TSI) in vitro lung model. Studies were then advanced to an in vivo lipopolysaccharide (LPS)-stimulated rodent model of inflammation. In parallel, the suitability of the siRNA-loaded nanoparticles for nebulization using a vibrating mesh nebuliser was assessed. The siRNA nanoparticles were nebulised using an Aerogen® Pro vibrating mesh nebuliser and characterised for aerosol output, droplet size and fine particle fraction. Only PEI anti-IL8 siRNA nanoparticles were capable of significant levels of IL-8 knockdown in vitro in non-nebulised samples. However, on nebulization through a TSI, only PEI-PEG siRNA nanoparticles demonstrated significant decreases in gene and protein expression in polarised Calu-3 cells. In vivo, both anti-CXCL-1 (rat IL-8 homologue) nanoparticles demonstrated a decreased CXCL-1 gene expression in lung tissue, but this was non-significant. However, PEI anti-CXCL-1 siRNA-treated rats were found to have significantly less infiltrating macrophages in their bronchoalveolar lavage (BAL) fluid. Overall, the in vivo gene and protein inhibition findings indicated a result more reminiscent of the in vitro bolus delivery rather than the in vitro nebulization data. This work demonstrates the potential of nebulised PEI-PEG siRNA nanoparticles in modulating pulmonary inflammation and highlights the need to move towards more relevant in vitro and in vivo models for respiratory drug development.

Highlights

Oligonucleotide therapeutics offer a unique opportunity for accurate and specific disease treatment at a genetic level
The siRNA sequences for human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin and IL-8 were obtained from Qiagen UK and were diluted 1:10 in TE buffer as directed
The PEI-LPEG was analysed via gel permeation chromatography (GPC), where it was found that the final product demonstrated a faster elution time than either the PEI or LPEG starting materials (Figure S1)

Summary

Introduction

Oligonucleotide therapeutics offer a unique opportunity for accurate and specific disease treatment at a genetic level To date, these have been investigated in a variety of endogenous and infectious conditions [1,2,3]. Local delivery is promising as an approach for oligonucleotide therapeutics targeting respiratory diseases This is especially true for episodes of acute pulmonary inflammation, including acute asthmatic episodes and pathogenic respiratory conditions such as acute respiratory distress syndrome (ARDS) [7,8]. COVID-19 is clinically manifest in the lungs in ~50% of patients and mortality is strongly linked to cytokine storm-induced ARDS [9,10,11] In these cases, a fast-acting specific treatment of short duration may be more favourable than a continuous, systemic depression of the innate immune system in an otherwise healthy individual. Anti-IL-8 therapy is currently under investigation in phase II clinical trials using i.v.-delivered anti-IL-8 monoclonal antibodies in COVID-19 patients [14]

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